Daphnia magna and Gammarus pulex, novel promising agents for biomedical and agricultural applications

Various studies have shown the importance of using different types of Zooplankton biomasses as an additional substance in the diet of fish. In addition, the drainage water of the fish cultures could be used in plant irrigation. In this study, biomasses of water flea Daphnia magna and Gammarus pulex collected and tested, for the first time, their effect against pathogenic microorganisms and on plant germination. The results showed significant antibacterial activity of D. magna and G. pulex against Staphylococcus aureus and Pseudomonas aeruginosa bacteria, as well as antifungal activity against Alternaria solani and Penicillium expansum, which gives the possibility to be used as biocontrol against these bacteria and plant pathogenic fungi. Furthermore, both animals showed positive activity in the germination rate of Vicia faba seed, reaching 83.0 ± 3.5 and 86.0 ± 3.8%, respectively. In conclusion, the biomasses of D. magna and G. pulex are promising and effective agents for their use in the medical field against some pathogenic microbes and as stimulators of plant growth.

usage of antibiotics and the deficiency of capabilities and scientific tools to improve such drugs had led scientists to explore alternative natural molecules with potential antimicrobial activity 25 .
Several types of bacteria and fungi can enter, individually or together, vital matrix called biofilm through a microbial-molecular communication system that regulates the virulence traits of these microbes and introducing infections by biofilm-forming microbes via medical devices 26 . The biofilm is a complex network of lipids and proteins used to protect microbes against a broad range of antibiotics and the host immune system [27][28][29] . Thus, it is necessary to search for new treatment substrates such as natural molecules to provide a new vision in treatment and limit the spread of MDR pathogens 28,[30][31][32] .
Different species of fungi cause the major common plant diseases, mostly controlled by synthetic fungicides 33 . Whereas repeated fungicide leads to the rapid evolution of fungal resistance against the fungicide, creating a high demand for exploring a natural alternative molecule 34 .
Broad beans or faba beans (Vicia faba) are considered one of the most important winter crops with high nutritional value, especially in Egypt 35 . The main fungal diseases infecting Vicia faba are wilting, root rot, leaf spot, and even plant death caused by species of diverse fungal genera such as Alternaria, Fusarium, Ascochyta, Colletotrichum, Rhizopus, Pythium, Rhizoctonia, and Clonostachys 36,37 . Hence, finding an eco-friendly substrate will be helpful in biocontrol plant diseases.
The discovery of natural antimicrobials might meet the consumer demand for environmentally friendly materials avoiding chemicals with harmful side effects 38,39 . There has been a promising trend for scientific research and industrial applications of biotechnology and marine compounds 40 . In this context, this study was conducted to studying for the first time the antibacterial, antifungal, and antibiofilm activities of the dried biomasses of G. pulex, and D. magna to be used safely in the fish diet and medical field, and limit the spread of some pathogenic bacterial and fungal microbes in addition to investigating the effect of these biomasses on plant seed germination. This report will open the door for further applications and valorization for other types of zooplankton as an inexpensive and safe alternative substrate.

Materials and methods
Tested animals. Daphnia magna and Gammarus pulex were collected from northern Egypt, Lake Mariout (31° 7.5′ N, 29° 47.1′ E) in June 2019; the salinity in this part of the lake is rather stable around 8‰ 39 .
Cladoceran species, Daphnia magna, and Amphipods, Gammarus spp., were collected with a standard plankton net of 200 µm mesh size, which was lowered vertically to a shallow bottom then pulled up to the water surface 5,7 . Then the net content was placed into plastic containers filled with lake water and identify D. magna and G. pulex through dissecting microscope. The samples were identified by traditional morphological and biochemical methods. The identified D. magna and G. pulex species were kept in 1001 transplant plastic holding containers that had dechlorinated tap water, with a salinity of 8% as in nature, for 24 h acclimatization before starting the cultivation experiments. After cultivation for a certain period under standard conditions, according to the methods described by Abo-Taleb et al. 11  Chemical analyses of the tested animals. The chemical composition of D. magna and G. pulex including moisture, carbohydrate, crude protein, fibers, crude fat, calories, and ash, was analyzed according to the standard methods of AOAC (1990) 6 . The moisture content was estimated by drying the samples to constant weight at 70 °C in a drying oven for 48 h. Whereas the nitrogen content was measured using an Automatic Kjeldahl system (UDK 139, VELP Scientifica). However, the lipid content was determined by ether extraction in multiunit extraction Soxhlet apparatus. The ash was determined by combusting dry samples in a muffle furnace at 550 °C for 3 h. Vitamins A, B2, B6, B12, D, E, and folic acid, besides the antioxidants such as tannic acid and β-carotene, have been determined in the cultivated D. magna and G. pulex to evaluate their nutritional value 5 .
Biological Activities of D. magna and G. pulex. Antibacterial and MIC determination. Antibacterial activities of D. magna and G. pulex were evaluated using an agar well diffusion assay on Muller-Hinton agar plates according to CLSI guidelines against human pathogenic bacterial strains Pseudomonas aeruginosa ATCC 27,853 and Escherichia coli ATCC8739, which represented Gram-negative bacteria, whereas Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 6538 were used to represent Gram-positive bacteria. The inhibition zone diameter was specified in millimeter (mm) 41 . The Micro-dilution method in micro-titer plates (MTP) was applied to determine the minimum inhibitory concentration (MIC) of active D. magna and G. pulex against tested pathogenic bacteria. Accordingly, D. magna and G. pulex were diluted in 1% DMSO at various concentrations (0.009-5.0 mg mL −1 , w/v) and tested against the pathogenic bacterial strains. Briefly, 1:100 (v/v) overnight cultures of the test strains were added to 200 µl of Mueller-Hinton broth media dispersed in MTP wells with/ without D. magna and G. pulex. Then, the plates were incubated with shaking at 120 rpm at 37 °C for 24 h. The lowest concentration of D. magna and G. pulex which inhibited bacterial growth was considered the MIC 42,43 . All experiments were performed in triplicates.
Antibiofilm assay. To analyze the anti-biofilm activity of the two animal species, a Microtiter plate (MTP) assay was used. D. magna and G. pulex were tested against two known biofilm-producing strains: Pseudomonas aeruginosa ATCC 27,853 and Staphylococcus aureus ATCC 6538. Sub-inhibitory concentrations (0.01-1.0 mg mL −1 ) of D. magna and G. pulex were loaded in a flat bottom MTP, containing Tryptic-Soy broth media (TSB) supplemented with 1% glucose and mixed well. The tested strains were cultured and incubated overnight in TSB at 37 °C and after incubation diluted to reach the turbidity of 1.5 × 10 8  www.nature.com/scientificreports/ tion period, the growth density was measured at OD 620 nm. Subsequently, the floated cells were transferred without troubling the formed biofilm and the plates were washed three times with sterilized phosphate-buffered saline (PBS) pH 7.4 to remove excess residue of floated unbounded cells. The biofilms in all wells were fixed with 200 µl of methanol 95% for 10 min. Then, crystal violet (CV 0.3% w/v) was added to each well using a multichannel micropipette (CAPP, Germany), and the plates were incubated for 15 min at room temperature. After that, the excess CV was removed, and the wells were gently washed with sterile distilled water. For the quantitative detection of biofilm formation, the adherent biofilm-bounded CV was assayed by eluting in 30% acetic acid and measured at OD 540 nm using an automated microplate reader (Tecan, Elx800-USA). The treated wells were compared with that of the untreated control. the untreated well inoculated with the test organism is considered the positive control while the negative control is the uninoculated media. All control and experiment groups were performed in triplicates.
The seed germinations test. were placed in distilled water in other Petri dishes. The germinating seeds were transferred to filter paper with 10 seeds per dish and a minimum distance of 1 cm between each seed (three replicates for each treatment). Distilled water was used for the control (untreated) experiments. For the germination rate and root growth investigation, the seeds were allowed to germinate for one week, then the seed germination percentage was calculated, and the seedling root length was measured. Four replicates were carried out for each treatment. Treated and control seeds were evaluated for record the percentage of seed germination with the following formula 46 : Statistical analysis. Three replicates were performed for each assay, and the standard error was calculated.
all resulting values were the averages of three independent replicates. The differences between a sample and the corresponding control were analyzed by using Student's t-test and the differences were considered significant if the p values were ≤ 0.05.
Ethical approval. This article does not contain any studies with human participants performed by any of the authors.

Results and discussions
Chemical analyses of tested animals. The chemical content of the two tested animals was analyzed to determine their prospective efficiency for industrial applications. Biochemical composition of D. magna and G. pulex is given in Table 1 based on three independent readings. The results point to higher chemical content of D. magna in protein, fibers, nitrogen-free extract, and both types of energy, while G. pulex had higher chemical content in fats, ash, and carbohydrate ( Table 1). The chemical composition of Gammarus can be affected by several factors such as age, season, habitats, region, and drying process after harvesting 21 (Fig. 1).
Therefore, the major constituents of D. magna and G. pulex biomasses were vitamin-A and β-carotene. The vitamin content in D. magna obtained from this study was lower than that mentioned by El-Feky et al. 2 , although the antioxidant contents were higher. On the other hand, the vitamin and antioxidant contents of G. pulex of this study showed a lower variation than mentioned by Abo-Taleb et al. 11 .

Germination(%) =
The number of germinated seeds The total number of used seeds ×  Table 2). The results indicated that G. pulex has better inhibitory effects against Gram-positive and Gram-negative pathogenic bacteria than D. magna. The inhibitory action for D. magna and G. pulex biomasses may be due to the reaction of the biomasses with bacterial protein by combining the thiol (-SH) group leading to the inactivation of proteins and bacterial growth. Therefore, it is necessary to test the MIC of D. magna and G. pulex for each bacterial strain ( Table 2). bioagents with lowest MIC values is candidate to be effective antimicrobial agents 48 . Results showed that the MIC for D. magna was 2.0 mg mL −1 against both P. aeruginosa and S. aureus. Moreover, MIC for G. pulex was 2.0 mg mL −1 against P. aeruginosa and S. aureus, respectively. Hence, biomasses have the same activity against Gram positive and negative bacteria. However, the mechanism of bacterial growth reduction through the interaction of D. magna and G. pulex still needs further studies.
The antibiofilm activity. In this work, the antibiofilm activity of the tested biomass exhibited varied effects against different bacterial strains (Fig. 2). Accordingly, G. pulex and D. magna biomass affected the biofilm     (Fig. 2a). On the other hand, the concentrations of 1, 0.5, and 0.25 mg mL −1 of G. pulex reduced the biofilm formation of P. aeruginosa by 90.38, 88.41, and 60.71% respectively, whereas the concentrations 1 and 0.5 mg mL −1 only affect the biofilm formation of S. aureus by 49.1 and 33.9% (Fig. 2b). The ability of these biomasses to reduce the biofilm formation of this opportunistic pathogens in a dose-dependent manner at sub-inhibitory concentrations motivating the researcher for more exploration to investigate the active compounds in these biomasses that may interfere with the biofilm formation pathway or the virulence regulation system in bacteria such as quorum sensing system. Our findings make these compounds promising molecules that could use as biofilm preventive agents. Interestingly, the primary stage of biofilm inhibition was observed at the MIC values 41 . The previous study explained the antibiofilm activity which showed deformation, external cell roughness, and cell wall shrinkage of bacterial cells 49 .
The activity against plant pathogenic fungi. This study investigated the activity of D. magna and G.
pulex biomasses against six plant pathogenic fungi (A. solani, A. niger, A. fumigatus, F. oxysporum, P. expansum, and R. solani). The results showed that D. magna and G. pulex had antifungal activity against two plant pathogenic fungi (A. solani and P. expansum) at 10 mg mL −1 ( Table 3). The inhibition zone diameters formed by the  (Fig. 3). D. magna enhanced the germination rate by 90, 83, and 80%, respectively (Fig. 3b), while with G. pulex the increase in germination rate was 86.6, 83, and 76.6%, respectively (Fig. 3c). Furthermore, D. magna showed high enhancement of root length with the highest effect recorded at 1 mg mL −1 of 222%, while G. pulex increased root length by 111% com- pulex on Vicia faba vegetative growth is that Chitosan reduced water uptake by minimizing plant transpiration. Moreover, nutrients, mineral vitamins, and antioxidants resulted in the regulation of Vicia faba metabolic processes, especially against stress conditions 57,58 . ‫‬

Conclusion
In this study, isolated biomass of D. magna and G. pulex showed obvious ability to inhibit several pathogens involving Gram-positive and Gram-negative bacteria, as well as inhibition against some plant pathogenic fungi. The current report considers the first biotechnological study of the current micro and macroinvertebrates. The biomass from D. magna and G. pulex exhibited the possibility of applying them in the medical and agricultural fields limiting the spread of some pathogenic microbes and enhancing plant germination.